Near-infrared light driven tissue-penetrating cardiac optogenetics via upconversion nanoparticles in vivo

Abstract

This study determines whether near-infrared (NIR) light can drive tissue-penetrating cardiac optical control with upconversion luminescent materials. Adeno-associated virus (AAV) encoding channelrhodopsin-2 (ChR2) was injected intravenously to rats to achieve ChR2 expression in the heart. The upconversion nanoparticles (UCNP) NaYF4:Yb/Tm or upconversion microparticles (UCMP) NaYF4 to upconvert blue light were selected to fabricate freestanding polydimethylsiloxane films. These were attached on the ventricle and covered with muscle tissue. Additionally, a 980-nm NIR laser was programmed and illuminated on the film or the tissue. The NIR laser successfully captured ectopic paced rhythm in the heart, which displays similar manipulation characteristics to those triggered by blue light. Our results highlight the feasibility of tissue-penetration cardiac optogenetics by NIR and demonstrate the potential to use external optical manipulation for non-invasive or weakly invasive applications in cardiovascular diseases.

Fig. 1.

ChR2-mCherry expression and electrophysiological characterization in cardiac optogenetics rat model. (a) Scheme of ChR2 virus vector delivery and 473-nm laser illumination for model verification. (b) ChR2 expressed in fusion with the mCherry fluorescence image in ventricles (red, ChR2; green, cardiomyocytes; blue, nuclei; scale bars, 50 µm). (c-g) Baseline ECG parameters in ChR2/mCherry, mCherry and control rats. P wave duration: ChR2/mCherry, 21.92 ± 0.77 vs. mCherry, 19.67 ± 3.27 vs. control, 21.86 ± 1.30, in ms; PR interval: ChR2/mCherry, 56.06 ± 4.07 vs. mCherry, 51.50 ± 5.46 vs. control, 56.72 ± 11.21, in ms; QRS duration: ChR2/mCherry, 19.31 ± 1.40 vs. mCherry, 20.03 ± 3.88 vs. control, 18.75 ± 1.44, in ms; QT interval: ChR2/mCherry, 61.89 ± 7.97 vs. mCherry, 56.03 ± 16.40 vs. control, 59.58 ± 8.59, in ms; n = 6; The data are presented as the mean ± SEM. NS, not significant, P > 0.05; one-way ANOVA/Bonferroni (c, e-g) and non-parametric Kruskal-Wallis test (d).

Fig. 2.

Electrophysiological characterization of cardiac optogenetics manipulations using the 473-nm laser in vivo. (a) Blue light pacing via illumination of different heart regions (blue line: 473 nm, 20 ms, 8 Hz, 0.96 mW/mm²; black line: ECG; RA: right atrium; LV: left ventricle; RV: right ventricle). (b-e) ECG parameters before, during, and after illumination of the right ventricle. QRS duration: light on, 32.67 ± 4.93 vs. before illumination, 19.08 ± 1.72, and vs. after illumination, 19.50 ± 1.96 in ms; PR interval: before illumination, 57.28 ± 3.59 vs. after illumination, 56.94 ± 4.53, in ms; QRS duration: before illumination, 19.08 ± 1.72, vs. after illumination, 19.50 ± 1.96 in ms; QT interval: before illumination, 69.11 ± 14.86 vs. after illumination, 71.64 ± 15.79 in ms; n = 6, The data are presented as the mean ± SEM. ****P < 0.0001; NS, P > 0.05; one-way ANOVA/Bonferroni (b, d, e) and paired t-test (c).

Fig. 3.

Properties of UCNP films and evaluations of NIR illumination. (a) Schematic diagrams of the procedure of 980-nm laser illumination via UCNP films. (b) TEM or FESEM images of 20 mg/ml UCNP and 200 mg/ml UCMP on the film (top left: TEM image of 20 mg/ml UCNP; top right: FESEM image of PDMS film; bottom left: FESEM image of 20 mg/ml UCNP on a PDMS film; bottom right: FESEM image of 200 mg/ml UCMP on a PDMS film).(c) Schematic ideogram of emission spectrum detection of the UCNP films. (d-f) Emission spectrum of the UCNP films at different concentrations under 980-nm power at 1 W (d) and 5 W (e), and upconversion intensity with elevated UCNPs concentrations (f). (g) Pulses NIR laser successfully captured right ventricle via 10 and 20-mg/ml UCNP films (red line: 980 nm, 20 ms, 8 Hz, 1.5 W; black line: ECG). (h) NIR power threshold via 10 and 20 mg/ml UCNP films respectively. n = 5; The data are presented as the mean ± SEM. NS, P > 0.05, unpaired t-test. (i) ECGs of failed NIR pacing via 2.5- and 5-mg/ml UCNP films.

Fig. 4.

Comparison of cardiac photoactivation using NIR laser via UCNPs and blue laser. (a) Emission spectrum of the 20-mg/ml UCNP film irradiated by a 980-nm laser from 1 to 5 W. (b, c) ECG (black line) triggered by two illumination methods on the right ventricles (b) red line: 980 nm, 20 ms, 8 Hz, 1.54 W; (c) blue line: 473 nm, 20 ms, 8 Hz, 0.19 mW/mm²)). (d, e) Illuminations on the right ventricle with pulse durations of 2, 5, 10, 20, and 50 ms with 980 nm (d) and 473 nm (E) laser. (f, g) Capture rate of the heart with increased power at 980 nm (f) and light intensity at 473 nm (g) with pulse durations of 2, 5, 10, 20, and 50 ms (n = 5; The data are presented as the mean ± SEM. *P < 0.05, ****P < 0.0001; NS P > 0.05; Chi-square test). (h, i) Minimum NIR laser power (h) and blue laser intensity (i) under different durations to achieve capture rate over 90% (n = 5; The data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, P > 0.05; one-way ANOVA/Bonferroni)). (j, k) Illuminations of the right ventricle with increasing pulse frequencies from 7 to 13 Hz under 980 (j) and 473 nm (k) laser. (l, m) Capture rates with increased pulse frequencies from 7 to 13 Hz with 980 and 473-nm lasers (l: 980 nm, 20 ms, 2.77 ± 0.79 W; m: 473 nm, 20 ms, 0.72 ± 0.18 mW/mm²; n = 5; The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01; Chi-square test).

Fig. 5.

Tissue-penetrating cardiac optogenetics activation by NIR via UCNPs or UCMPs. (a) Schematic diagram of tissue-penetrating manipulation by NIR. (b) Emission spectrum of 20-mg/ml UCNP film and 200-mg/ml UCMP film illuminated under an NIR power of 1 W. (c, d) ECG recording (c) and determination of NIR (8 Hz, 20 ms) power threshold (d) of cardiac capture by NIR through different tissue thicknesses on 20-mg/ml UCNP film. (e, f) ECG recording (e) and determination of NIR power threshold (f) of cardiac capture by NIR through different tissue thicknesses via 200-mg/ml UCMP film. The data are presented as the mean ± SEM.